Recognition and killing of cancer stem-like cell population in hepatocellular carcinoma cells by cytokine-induced killer cells via NKG2d-ligands recognition.

There is an urgent need for more potent and safer approaches to eradicate cancer stem cells (CSCs) for curing cancer. In this study, we investigate cancer-killing activity (CKA) of cytokine-induced killer (CIK) cells against CSCs of hepatocellular carcinoma (HCC). To visualize CSCs in vitro by fluorescence imaging, and image and quantify CSCs in tumor xenograft-bearing mice by bioluminescence imaging, HCC cells were engineered with CSC detector vector encoding GFP and luciferase controlled by Nanog promoter. We found that CIK cells have a strong CKA in vitro against putative CSCs of HCC, as shown by tumorsphere formation and time-lapse imaging. Additionally, time-lapse recording firstly revealed that putative CSCs were attacked simultaneously by many CIK cells and finally eradicated by CIK cells, indicating the necessity of achieving sufficient effector-to-target ratios. We firstly illustrated that anti-NKG2D antibody blocking partially but significantly inhibited CKA of CIK cells against putative CSCs. More importantly, intravenous infusion of CIK cells remarkably delayed tumor growth in mice with a significant decrease in putative CSC number monitored by bioluminescence imaging. Taken together, these findings demonstrate CKA of CIK cells against putative CSCs of HCC, at least in part, by NKG2D-ligands recognition.

[Changes in lymphocyte subsets in infants with common lower respiratory tract infectious diseases].

OBJECTIVE: To investigate the changes and clinical significance of lymphocyte subsets in infants with bronchitis, bronchopneumonia, and bronchiolitis. METHODS: A total of 111 children with bronchitis, 418 children with bronchopneumonia, and 83 children with bronchiolitis were enrolled as disease groups, and 235 healthy children were enrolled as control group. Flow cytometry was applied to measure lymphocyte subsets. RESULTS: The bronchitis group had significantly lower numbers of T cells and CD3+CD8+ T cells than the control group (P<0.05). The bronchopneumonia group had significantly lower numbers of T cells and CD3+CD8+ T cells, a significantly higher number of T helper (Th) cells, and a significantly higher CD4/CD8 ratio than the control group, as well as a significantly higher number of Th cells than the bronchitis group. Compared with the children with mild bronchopneumonia, those with severe bronchopneumonia showed a reduction in T cells and an increase in B cells (P<0.05). The bronchiolitis group had a significantly higher number of Th cells, a significantly higher CD4/CD8 ratio, and a significantly lower number of CD3+CD8+ T cells than the control group (P<0.01). The disease groups showed a significantly higher number of B cells and a significantly lower number of natural killer cells than the control group (P<0.05). CONCLUSIONS: A low, disturbed cellular immune function and a high humoral immune function are involved in the development and progression of lower respiratory tract infectious diseases. The changes in immune function are related to the type and severity of diseases.

Cytokine-induced killer cells efficiently kill stem-like cancer cells of nasopharyngeal carcinoma via the NKG2D-ligands recognition.

Cancer stem cells (CSCs) are considered to be the root cause for cancer treatment failure. Thus, there remains an urgent need for more potent and safer therapies against CSCs for curing cancer. In this study, the antitumor activity of cytokine-induced killer (CIK) cells against putative CSCs of nasopharyngeal carcinoma (NPC) was fully evaluated in vitro and in vivo. To visualize putative CSCs in vitro by fluorescence imaging, and image and quantify putative CSCs in tumor xenograft-bearing mice by in vivo bioluminescence imaging, NPC cells were engineered with CSC detector vector encoding GFP and luciferase (Luc) under control of Nanog promoter. Our study reported in vitro intense tumor-killing activity of CIK cells against putative CSCs of NPC, as revealed by percentage analysis of side population cells, tumorsphere formation assay and Nanog-promoter-GFP-Luc reporter gene strategy plus time-lapse recording. Additionally, time-lapse imaging firstly illustrated that GFP-labeled or PKH26-labeled putative CSCs or tumorspheres were usually attacked simultaneously by many CIK cells and finally killed by CIK cells, suggesting the necessity of achieving sufficient effector-to-target ratios. We firstly confirmed that NKG2D blockade by anti-NKG2D antibody significantly but partially abrogated CIK cell-mediated cytolysis against putative CSCs. More importantly, intravenous infusion of CIK cells significantly delayed tumor growth in NOD/SCID mice, accompanied by a remarkable reduction in putative CSC number monitored by whole-body bioluminescence imaging. Taken together, our findings suggest that CIK cells demonstrate the intense tumor-killing activity against putative CSCs of NPC, at least in part, by NKG2D-ligands recognition. These results indicate that CIK cell-based therapeutic strategy against CSCs presents a promising and safe approach for cancer treatment.

Oxygen free radicals and mitochondrial signaling in oligospermia and asthenospermia.

The aim of this study was to investigate the roles of oxygen free radicals and mitochondrial signaling in semen disorders, in particular, how this induces low concentrations and reduced motility of sperm. Ejaculate samples were obtained from 120 young adult males (mean age, 28.7±5.3 years) with normal semen (n=30), oligospermia (n=30), asthenospermia (n=30) and oligoasthenozoospermia (n=30). The malondialdehyde (MDA) content, total superoxide dismutase (T­SOD) activity and glutathione peroxidase (GSH­Px) activity of the sperm samples were determined by enzymatic assays. Mitochondrial membrane potential (MMP) was determined by flow cytometric detection of accumulated membrane­permeable JC­1 fluorescent dye. The mRNA and protein expression levels of apoptosis-associated genes [Bcl­2, Bax, cytochrome c (Cyt C) and caspase-3] were measured by quantitative polymerase chain reaction and western blotting. Intergroup differences were evaluated by Student's t­test. The sperm samples from all semen disorder groups exhibited significantly lower T­SOD content and GSH­Px activity (all P<0.05 versus normal sperm), and the extent of reduction revealed a disorder-associated trend (asthenospermia < oligospermia ≈ oligoasthenozoospermia). By contrast, the semen disorder groups had significantly higher MDA content (all P<0.05 versus normal sperm); the extent of this increase also revealed a disorder-associated trend (asthenospermia > oligospermia ≈ oligoasthenozoospermia). The sperm from patients with semen disorders also exhibited significantly lower MMP than normal sperm, as evidenced by lower mean ratios of JC­1+ sperm per group. The semen disorder groups had significantly higher Bax, Cyt C and caspase-3 mRNA and protein expression levels in the sperm samples, but significantly lower levels of Bcl­2 (all P<0.05 versus normal sperm). However, only the extent of increases in Cyt C and caspase-3 exhibited a disorder-associated trend (oligospermia > asthenospermia). In conclusion, oxygen free radicals may be involved in reduced sperm concentration and motility, possibly through effects on the mitochondrial apoptotic signaling pathway. Perturbed mitochondrial release of Cyt C may be the distinguishing molecular feature between oligospermia and asthenospermia.

[Human leukocyte antigen-G 14 bp deletion polymorphism in severe pre-eclampsia].

OBJECTIVE: To investigate the relationship between human leukocyte antigen-G (HLA-G) gene Exon 8 14 bp deletion polymorphism and the pathogenesis of severe pre-eclampsia. METHODS: Forty-two pregnant women with severe pre-eclampsia, who admitted to the Third Affiliated Hospital of Zhengzhou University from October 2008 to February 2009, and their newborns were chosen as the severe pre-eclampsia group. Another 45 healthy gravidas at the third trimester and their newborns were chosen as the control. All gravidas in both groups were Han Nationality. HLA-G Exon 8 genotyping was detected by PCR in both groups and the allele frequencies and genotype frequencies were compared between the two groups. The genotype frequencies of maternal-neonatal pairs were also analyzed. RESULTS: (1) In the severe pre-eclampsia group, 14% of the maternal-neonatal pairs were homozygote of 14 bp deletion, and significantly higher frequency 33% (15/45) was found in the control group (P=0.038). (2) No significant difference was found in the allele frequencies and genotype frequencies of HLA-G 14 bp deletion polymorphism among all the mothers between the two groups (P>0.05). (3) The +14 bp and -14 bp allele frequencies of HLA-G 14 bp deletion polymorphism in newborns in the severe pre-eclampsia group were 44% (37/84) and 56% (47/84), respectively, and 30% (27/90) and 70% (63/90) in the control group. Although there was no significant difference between the two groups, but differences in trends was identified (chi2=3.678 P=0.055); The genotype (-14 bp/-14 bp) frequency of neonates in the severe pre-eclampsia group showed no difference compared with that in the control group [29% (12/42) vs 49% (22/45)], but differences in trends was also found (P=0.052). CONCLUSIONS: HLA-G 14 bp deletion polymorphism is associated with the susceptibility of severe pre-eclampsia in Chinese Han nationality. Maternal-fetal genotype pairs of -14 bp/-14 bp may have reduced risk of severe pre-eclampsia.

Polymorphism of HLA-A and HLA-B in pre-eclampsia.

OBJECTIVE: To investigate the association between the polymorphism of HLA-A, HLA-B genes and pre-eclampsia. METHODS: HLA-A, HLA-B genotyping was performed by polymerase chain reaction sequence-specific primer (PCR-SSP) in 119 preeclampsia patients, 117 normal pregnant women and their neonates. RESULTS: The study showed that 16 HLA-A and 39 HLA-B alleles were obtained in pre-eclamptic patients and normal pregnant women. 15 HLA-A and 37 HLA-B alleles were obtained in their neonates. No significant difference was found in maternal or neonatal HLA-A, HLA-B alleles between pre-eclampsia group and control group (P(c) > 0.05). The frequencies of HLA-A11, HLA-A24, HLA-B13, HLA-B14, HLA-B15, HLA-B52 maternal/fetus genetic associations were significantly different between pre-eclampsia group and control group (P < 0.05). CONCLUSION: Some HLA-A,HLA-B maternal/fetus special bindings may be associated with the susceptibility or protective of pre-eclampsia.